Methods for the manufacture of proteolytically processed polypeptides

Abstract

A proteolytically active polypeptide comprising an acid sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1 with the proviso that the polypeptide does not comprise the amino acid sequence of SEQ ID NO: 1. A nucleic acid encoding the polypeptide. An antibody specifically binding to the polypeptide. A method for the manufacture of a proteolytically processed polypeptide comprising contacting a first polypeptide having at least 50% sequence identity to SEQ ID NO: 1 with a second polypeptide that is susceptible to proteolysis by said first polypeptide.

The invention claimed is: 
     
       1. A method for producing a proteolytically processed polypeptide, the method comprising contacting a first polypeptide having at least 50% sequence identity with SEQ ID NO: 1 with a second polypeptide that is susceptible to proteolysis by the first polypeptide, resulting in the proteolytic processing of the second polypeptide into at least two cleavage products; wherein: the method occurs in vitro; and/or at least one of the first or second polypeptide is recombinant or purified. 
     
     
       2. The method of  claim 1 , wherein the second polypeptide comprises an amino acid sequence having at least 50% sequence identity swill any one of SEQ ID NOs: 3 to 25. 
     
     
       3. The method of  claim 1 , wherein the second polypeptide is a clostridial neurotoxin. 
     
     
       4. The method of  claim 1 , wherein the second polypeptide is selected from: a clostridial neurotoxin polypeptide that lacks a functional binding domain (H cc ), a clostridial neurotoxin polypeptide that has a modified clostridial neurotoxin binding domain (H cc ) that binds to a native clostridial neurotoxin receptor; or a clostridial neurotoxin polypeptide having a non-clostridial binding domain that binds to a non-native clostridial neurotoxin receptor. 
     
     
       5. The method of  claim 4 , wherein the clostridial neurotoxin comprises a light chain from a first clostridial neurotoxin serotype and/or sub-type and a heavy chain from a second and different clostridial neurotoxin serotype and/or subtype. 
     
     
       6. The method of  claim 1 , wherein the second polypeptide is a single-chain clostridial neurotoxin prepared by recombinant expression in  E. coli.    
     
     
       7. The method of  claim 1 , wherein the first polypeptide is purified and has 100% sequence identity to SEQ ID NO: 1. 
     
     
       8. The method of  claim 1 , wherein the second polypeptide recombinant. 
     
     
       9. The method of  claim 1 , wherein die second polypeptide is recombinant BoNT/A. 
     
     
       10. A method for proteolytically processing a polypeptide in a subject wherein a first polypeptide having at least 50% sequence identity with SEQ NO: 1 and a second polypeptide that is susceptible to proteolysis by the first polypeptide are both administered to a subject and allowed to contact each other, resulting in the proteolytic processing of the second polypeptide into at least two cleavage products. 
     
     
       11. The method of  claim 1 , wherein the First and second polypeptides are incubated together for at least 30 minutes. 
     
     
       12. The method of  claim 9 , wherein the first polypeptide has at least 85% sequence identity with SEQ ID NO: 1. 
     
     
       13. The method of  claim 1 , wherein the first polypeptide has at least 90% sequence identity with SEQ ID NO: 1. 
     
     
       14. The method of  claim 1 , wherein the first polypeptide has at least 95% sequence identity with SEQ ID NO: 1. 
     
     
       15. The method of  claim 1 , wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID NO: 1 by up to 50 conservative amino acid exchanges. 
     
     
       16. The method of  claim 1 , wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID NO: 1 by up to 20 conservative amino acid exchanges. 
     
     
       17. The method of  claim 1 , wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID NO: 1 by up to 10 conservative amino acid exchanges. 
     
     
       18. The method of  claim 10 , wherein the first polypeptide has at least 95% sequence identity with SEQ ID NO: 1. 
     
     
       19. The method of  claim 10 , Wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID No: 1 by up to 20 conservative amino acid exchanges. 
     
     
       20. The method of  claim 10 , wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID NO: 1 by up to 10 conservative amino acid exchanges.